Ion exchangers

ABSTRACT

The present invention provides a new quaternary amino (QA) anion exchanger comprising QA derivatised, hydroxy (C2-C4) alkylated and cross-linked regenerated cellulose, in which the level of derivatisation with the QA-groups is 1.4 meg/g or greater.

FIELD OF THE INVENTION

[0001] This invention relates to new cellulosic ion exchangers usefulfor separating proteins from protein-containing solutions, andparticularly for separating whey proteins from whey protein-containingsolutions. The invention also relates to processes for preparing the ionexchangers.

BACKGROUND OF THE INVENTION

[0002] Ion exchangers have been used for many years to separate outproteins from protein-containing solutions. They have found applicationin the dairy industry, particularly in recovering whey proteins frommilk and milk derived process streams, such as whey and whey proteinconcentrates.

[0003] Ion exchangers which have been used in separating whey proteinsfrom whey protein containing solutions include both cation exchangers,particularly of the SP or SE (sulphonate) or CM (carboxymethyl) type,and anion exchangers, particularly of the QA (quaternary amino) or DEAE(diethylaminoethyl) type. In terms of the exchanger matrix itself, manyinsoluble matrices have been used, including cellulose, cross-linkeddextran, cross-linked agarose, synthetic hydophilic polymers andinorganic materials coated with hydrophilic polymers.

[0004] One matrix that has proved to be particularly useful in largescale separation and purification of whey proteins is regeneratedcellulose which has been hydroxyalkylated and cross-linked. Ionexchangers prepared on this matrix are resistant to attrition, have highprotein capacity, high flow properties and are available at relativelylow cost.

[0005] Examples of such ion exchangers based on a hydroxyalkylated andcross-linked regenerated cellulose matrix which are commerciallyavailable include the SP, CM, QA, and DEAE exchangers sold as SPGibcoCel™, CM GibcoCel™, QA GibcoCel™ and DEAE GibcoCel™ respectively.These ion exchangers were previously sold under the Indion™ brand name.QA GibcoCel™ and SP GibcoCel™ having a substitution level of the QA orSP groups of up to 1.2 milli-equivalents per dry gram (meq/g) areavailable. SP GibcoCel™, a cation exchanger, has been widely used, butQA GibcoCel™, an anion exchanger, has only enjoyed limited useindustrially.

[0006] Levison et al (Chimica Oggi/Chemistry Today, 41-48, Nov/Dec 1994)refers to three custom made QA celluloses with substitution levels of0.74, 0.96 and 1.24 meq/g, and discloses that these had similar proteincapacities.

[0007] Anion exchangers bearing quaternary amino (QA) groups aretypically made by alkylation of either a hydrophilic hydroxyl-bearingmatrix or such a matrix already bearing tertiary amino groups such asdiethylaminoethyl (DEAE) groups. In the latter case simple alkylatingagents may be used such as ethylene oxide as shown in the followingequation.

[0008] Direct alkylation of the hydrophilic matrix is achieved usingagents already containing a quaternary ammonium group, eg

[0009] Several such reagents are summarized in U.S. Pat. No. 5,731,259and many of them are available commercially for large scale industrialuse.

[0010] Alkylating agents (3-chloro-2-hydroxypropyl)trimethylammoniumchloride (CHPTAC) and glycidyltrimethylammonium chloride (GTAC) havebeen widely used to prepare quaternary ammonium derivatives (cationicderivatives) of polysaccharides, especially starch and cellulose. Bothwater soluble and water insoluble derivatives have been prepared for avariety of purposes. Only the latter are useful as anion exchangers forthe adsorption and chromatography of proteins.

[0011] Japanese patent 79042385 (1979) and Chemical Abstracts 91, 58084(Toyo Pulp KK) describe the preparation of a crosslinked QA cellulosewith a degree of substitution (DS) of 0.13 (<<1 meq/g) and a proteincapacity of 0.24 g/g, using 50% CHPTAC.

[0012] CS 202,374 (1983) and Chemical Abstracts 99, 72429 describe thepreparation of ion exchangers with capacities of 0.37 to 0.68 meq/g frompowdered cellulose. Analogous products were also obtained fromcrosslinked cellulose, hydroxyethyl cellulose and starch and stated tobe useful as ion exchangers, sorbents and flocculants.

[0013] A further Czechoslovakian patent, CS 236,024 (1987), and ChemicalAbstracts 109, 151774 describe the preparation oftrimethylammoniumhydroxypropyl cellulose, an ion exchanger with anexchange capacity of 0.24 meq/g after first activating the cellulosewith acetic or phosphoric acid.

[0014] Several 1989 Japanese patents, JP 01/130,726; 01/106,898 and01/099,646 (Daicel Chemical Industries, Ltd) (Chemical Abstracts 112,95049; 112, 135584 and 113, 20528) disclose the preparation ofcrosslinked, cationized hydroxy-alkylcellulose gels for chromatographyof nucleic acids. For example, hydroxyethyl cellulose is reacted withGTAC or CHPTAC and crosslinked and used to bind nucleic acidsselectively from a mixture of nucleic acids and proteins. Lowsubstitution levels of QA groups are typically useful for bindingnucleic acids but not proteins, hence the selectivity observed.

[0015] WO 91/17830 describes the use of regenerated cellulose to preparea crosslinked flexible sponge with fibrous reinforcement. This was thenderivatized by reaction with CHPTAC to give a QA cellulose sponge with aprotein binding capacity of 1.5 g/g. Such products have yet to be madeand demonstrated on the very large scale needed for use in the dairyindustry.

[0016] Antal et. al. (Carbohydrate Polymers 19, 167-169, 1992) describethe optimization of the reaction of microcrystalline cellulose with thealkylating agents CHPTAC and1,3-bis(3-chloro-2-hydroxy-propyl)imidazolium hydrogen sulfate inalkaline medium. The maximum substitution level they were able to obtainwith CHPTAC was 0.94 meq/g (mmol/g), although the second reagent gave aproduct with 1.56 meq/g. No protein capacities are given and it islikely that the latter reagent, being bifunctional, would haveintroduced extensive crosslinking into the cellulose to the detriment ofprotein capacity. Furthermore microcrystalline cellulose is not asuitable matrix for repeated use on a large industrial scale.

[0017] CHPTAC has been used to make a bead-shaped QA starch anionexchanger with exchange capacity of 0.90 meq/g. (Chemical Abstracts 130,63153, 1998). The corresponding diethylaminoethyl (DEAE) starch madeusing 2-chloroethyl(diethyl)amine hydrochloride had a capacity of 2.47meq/g showing the greater difficulty typically experienced in making thequaternary amino (QA) derivatives than for the tertiary aminoderivatives like DEAE.

[0018] Fibrous cellulose has been derivatized with quaternary ammoniumgroups to a high degree of substitution, DS of at least 0.5 (>2 meq/g),using a very large excess of alkylating reagent containing quaternaryammonium groups. The cellulose is either not crosslinked (1998 U.S. Pat.No. 5,731,259) or crosslinked (1998 U.S. Pat. No. 5,780,616). Preferablythe alkylating reagent is used in 20:1 to 40:1 mole ratio of reagent toanhydroglucose units of cellulose. In the case of GTAC this amounts to186-372 g of reagent per 10 g of cellulose used either in 5-8 repeatedreactions or one large addition of the solid reagent with 30 mL ofwater. The products, described at one point as a jelly mass, are usefulas superabsorbents for water and saline solutions in the field ofhygenic-sanitary products such as diapers for babies. They are designedto be used once and then disposed of and are not at all suitable forrepeated use day after day in a reactor or column bed where physicalrobustness against attrition, long life and high flow-through rates arerequired for anion exchangers processing protein solutions.

[0019] With the above background in mind, it was an object of thepresent invention to provide an anion exchanger which is particularlyuseful on an industrial scale in separating whey proteins from wheyprotein containing solutions, or at least to provide the public with auseful choice.

SUMMARY OF THE INVENTION

[0020] Accordingly, in a first aspect the present invention provides ananion exchanger comprising a water insoluble, hydrophilic, waterswellable, hydroxy(C₂-C₄) alkylated and cross-linked regeneratedcellulose, derivatised with quaternary amino (QA) groups, wherein thelevel of substitution of the QA groups is 1.4 milliequivalents per drygram of anion exchanger (meq/g) or greater.

[0021] Preferably, the level of substitution of QA groups is from about1.4 to about 2.5 meq/g, more preferably from about 1.5 to about 2.5meq/g, and most preferably from about 1.7 meq/g to about 2.5 meq/g.

[0022] Preferably, the cellulose is hydroxypropylated cross-linkedregenerated cellulose.

[0023] In a further aspect, the present invention provides a process ofpreparing an anion exchanger as defined above, the process comprisingthe step of reacting an anion exchanger comprising a water-insoluble,hydrophilic, water swellable, hydroxy(C, 2-C₄)alkylated and cross-linkedregenerated cellulose derivatised with quaternary amino (QA) groups,wherein the level of substitution of QA groups is less than 1.4 meq/g,with an alkylating agent or agents capable of derivatising the anionexchanger with QA groups, under conditions suitable to achieve a levelof substitution of QA groups of 1.4 meq/g or greater on the anionexchanger.

[0024] In a further aspect, the present invention provides a process ofpreparing an anion exchanger as defined above, the process comprisingthe step of reacting a water-insoluble, hydrophilic, water swellable,hydroxy(C₂-C₄)alkylated and cross-linked regenerated cellulose with analkylating agent capable of derivatising the cellulose with QA groups,under conditions suitable to derivatise the cellulose and achieve alevel of substitution of QA groups of 1.4 meq/g or greater. Optionally,the above process may include the additional step of further reactingthe QA-derivatised anion exchanger thus prepared with an alkylatingagent capable of derivatising the anion exchanger with QA groups toachieve a higher level of substitution of QA groups.

[0025] Preferably, the alkylating agent is(3-chloro-2-hydroxypropyl)trimethylammonium chloride (CHPTAC).

[0026] Alternatively, the alkylating agent is glycidyltrimethylammoniumchloride (GTAC).

[0027] In a further aspect, the present invention provides an anionexchanger obtainable by a process as defined above.

[0028] While the invention is broadly as defined above, it is notlimited thereto and also includes embodiments of which the followingdescription provides examples.

DESCRIPTION OF THE DRAWINGS

[0029] The present invention will now be described in more detail. Inparticular, a better understanding of the invention will be gained withreference to the accompanying drawings, in which:

[0030]FIG. 1 shows the QA substitution level as a function of the volumeof 50% GTAC reagent used to prepare QA-HP cellulose samples inComparative Example 1;

[0031]FIG. 2 shows the protein capacity as a function of the QAsubstitution level for the QA-HP cellulose samples of ComparativeExample 1;

[0032]FIG. 3 shows the protein capacity as a function of NaClconcentration for the anion exchanger QA GibcoCel™ and for an anionexchanger according to the present invention (QA cellulose #2 (1.84meq/g)); and

[0033]FIG. 4 shows the protein capacity as a function of NaClconcentration for the anion exchanger QA GibCoCel™ and two anionexchangers according to the present invention (QA cellulose 2.08 meq/gand 2.52 meq/g).

DESCRIPTION OF THE INVENTION

[0034] As defined above, the present invention relates to new quaternaryamino (QA) anion exchangers. In particular, the anion exchangers of thepresent invention comprise QA derivatised, hydroxy(C₂-C₄)alkylated andcross-linked regenerated cellulose, in which the level of derivatisationwith the QA groups is 1.4 meq per dry gram of anion exchanger (meq/g) orgreater.

[0035] The applicants have now found that it is possible to prepare QAderivatives of hydroxyalkylated regenerated cellulose havingsubstitution levels higher than those described in the prior art. Wehave also surprisingly found that such derivatives, where they exceedsubstitution levels of 1.4 meq/g, possess advantages over QA derivativesof hydroxylated cross-linked regenerated cellulose having lowersubstitution levels, in that they have a significantly higher effectiveprotein binding capacity when used to recover protein fromprotein-containing solutions having more than a relatively low ionicstrength, and in particular milk protein-containing solutions such aswhey and whey protein concentrates.

[0036] The finding that a substituted QA-derivatised anion exchanger ofthe GibcoCel™ type having a minimum substitution level of 1.4 meq/g hasan improved protein binding capacity over currently available QA anionexchangers (which have a level of derivatisation of up to 1.2 meq/g) forsolutions such as whey is particularly surprising, in view of the factthat the corresponding SP cation exchangers having 0.8 and 1.4 meq/ghave been found to be almost equally effective at adsorbing protein fromwhey (Ayers & Peterson N.Z. J. Dairy SCL and Technol., 20, 129-142,1985).

[0037] It is these findings by the applicants which form the basis ofthe present invention.

[0038] In this specification, the term “QA” or “quaternary amino”, whenused in the context of ion exchangers, means a functional group selectedfrom a group of the formula —R₁-Z, wherein R₁ is a lower alkylene groupcontaining 1 to 3 carbon atoms and optionally substituted with ahydroxyl group, and Z is a quaternized amino group of the formula:—NR₂R₃R₄ ⁺OH⁻ or salts thereof, wherein R₂, R₃ and R₄ are each a loweralkyl group containing 1 to 4 carbon atoms, optionally substituted witha hydroxyl group, or a further group of the formula —R₁—NR₂R₃R₄ ⁺OH⁻ orsalts thereof wherein R₁, R₂, R₃ and R₄ are as defined above. Examplesof suitable QA groups are —CH₂CH₂N⁺R₂R₃R₄Cl⁻ and —CH₂CHOHCH₂N⁺R₂R₃R₄Cl⁻,wherein R₂, R₃ and R₄ are the same or different and are selected from—CH₃, —CH₂CH₃, —CH₂CH₂OH, —CH₂CHOHCH₃, —CH₂CH₂N⁺R₂R₃R₄Cl⁻ and—CH₂CHOHCH₂N⁺R₂R₃R₄Cl⁻.

[0039] It is preferred that in the QA anion exchangers of the presentinvention, the level of substitution of the QA groups is in the range offrom about 1.4 to about 2.5 meq/g, more preferably from about 1.5 toabout 2.5 meq/g, and most preferably from about 1.7 meq/g to about 2.5meq/g.

[0040] The matrix for the anion exchangers of the present inventioncomprises a water insoluble, hydrophilic, water swellablehydroxy(C₂-C₄)alkylated and cross-linked regenerated cellulose. Suchmatrices and processes for preparing them are described for example inU.S. Pat. No. 4,175,183 (John S Ayers), the full contents of which areincorporated herein by reference. By way of example, a suitablecellulose matrix can be prepared by reacting commercially availablegranular or beaded regenerated cellulose with epichlorohydrin andpropylene oxide in the presence of a strong base (conveniently NaOH).Such matrices may be useful for repetitive use on a large industrialscale.

[0041] The QA anion exchangers of the present invention having asubstitution level of 1.4 meq/g or greater may be prepared by reacting acellulose matrix as described above with a suitable alkylating agentcapable of derivatising the cellulose with QA groups. Conveniently, thealkylating agent may be an agent containing quaternary ammonium groups,preferably (3-chloro-2-hydroxypropyl)trimethylammonium chloride (CHPTAC)or glvcidyltrimethyl ammonium chloride (GTAC), and the reaction carriedout in the presence of a strong base, conveniently sodium or potassiumhydroxide. CHPTAC and GTAC are known reagents for introducing QA groupsinto cellulose, but at lower substitution levels (see, for exampleCarbohydrate Polymers 19, 167-169, 1992). However, in order to preparethe anion exchangers having the level of QA substitution of the presentinvention, it will usually be necessary to employ very concentratedsolutions of reagents. For example, it is preferred that when CHPTAC isused as the alkylating agent, the concentration of CHPTAC reagent isabout 50 wt % or greater, more preferably about 60 wt % or greater, andwhen GTAC is used, the concentration of GTAC reagent is greater than 50%w/v, more preferably about 70% w/v or greater.

[0042] By way of example, the following process may be used to preparethe QA anion exchangers of the present invention, having a substitutionlevel of 1.4 meq/g or greater.

[0043] A water insoluble, hydrophilic, water swellable hydroxypropylatedand cross linked regenerated cellulose may be prepared by first forminga mixture of 10 g regenerated cellulose with 3-10 mL of propylene oxide,0.5-1 mL of epichlorohydrin and 8-20 mL of aqueous sodium hydroxidesolution at a concentration of 15-40% (w/v), or 10-15 mL of aqueoussodium hydroxide solution at a concentration of 20-30% (w/v). Themixture is then reacted at 40-60° C. for 1-4 hours. At the end ofreaction, most of the sodium hydroxide remains in the matrix still, asonly the reactions of epichlorohydrin consume base. It is preferable toleave the hydroxide in the cellulose for further reaction with thealkylating reagent.

[0044] When GTAC is used as the alkylating agent, 15-20 mL of 70% (w/v)solution is mixed in with the cellulose and reaction accomplished at10-50° C. over 1-8 hours, preferably 20-25° C. for 2-3 hours. Thereaction of GTAC is catalysed by hydroxide. The reaction time andtemperature are thus not greatly dependent on the amount of GTAC added.The amount of GTAC reagent added will be selected to achieve the desiredsubstitution level.

[0045] When CHPTAC is used as the alkylating agent, 12-20 mL of a 60 wt% solution is mixed in with the cellulose and reaction accomplished at20-50° C. for 2-24 hours, preferably at 25° C. for 6-24 hours, althoughthe time can be shortened by heating to 60-80° C. for 1-2 hours at thefinish. Hydroxide is consumed during this reaction and the reaction timeincreases as the amount of reagent used is increased as a result ofconsumption of hydroxide by the competing alkylation and hydrolysisreactions.

[0046] In the case of both the GTAC and CHPTAC reagents it is preferableto keep the volume added to 20 mL or less (when working with the aboveproportions) so that a separate aqueous phase does not separate out fromthe cellulose. To limit the competing hydrolysis reactions of thereagents it is desirable to limit the amount of water present in thereaction mixture and use the highest concentration of reagent available.

[0047] In either of the above processes, it is possible to repeat the QAderivatisation procedure, if required, where a relatively high level ofderivatisation (such as around 2.0 meq/g or higher) is desired. In suchcases the procedure described in the following alternative embodimentswould be used.

[0048] In an alternative embodiment, the QA anion exchangers of thepresent invention may be prepared by using as the starting material acommercially available QA hydroxyalkylated and cross-linked regeneratedcellulose, such as that sold as QA GibcoCel™, which has a QAsubstitution level of 1.2 meq/g. The applicants have found that a higherQA substitution level can be achieved by further processing the alreadyderivatised exchanger using alkylating agents bearing quaternaryammonium groups such as GTAC and CHPTC, again in the presence of astrong base.

[0049] By way of example, the following process may be used to preparethe QA anion exchangers of the present invention, having a substitutionlevel of 1.4 meq/g or greater, using a similarly but lower substitutedexchanger (conveniently QA GibcoCel™) as a starting material.

[0050] QA GibcoCel™ in its hydrated form has a dry matter content ofonly 12-13%. Because of the large amount of water already present in theproduct, it is often preferable to process this further as a slurry withalkylating agent and base. Accordingly, hydrated QA GibcoCel™ is mixedwith extra water and concentrated sodium hydroxide solution to form athick slurry with a final sodium hydroxide concentration of 1.5-3.0%(w/v), taking into account the water already present in the hydrated QAGibcoCel™ (85-90% of its wet weight). CHPTAC, at a concentration of 60wt %, is added in an amount of 5-25 mL/100 g of QA GibcoCel™ to achievethe desired increase in substitution level. The conditions should bechosen such that there is an excess of hydroxide present over CHPTAC inthe reaction mixture. Reaction is generally accomplished at 10-50° C.for 2-24 hours, preferably 20-30° C. for 6-24 hours, or 17 hours with afurther 1-2 hours at 60-80° C.

[0051] In either of the above methods of preparing a QA anion exchangerof the invention, the sodium hydroxide could be replaced by anequivalent amount of potassium hydroxide.

[0052] As mentioned above, the applicants have found that the anionexchangers of the present invention have a higher protein capacity underall conditions of ionic strength, except low ionic strength (eg <25 mM)than known, commercially available QA derivatised hydroxyalkylatedcross-linked regenerated cellulose anion exchangers with a lower levelof QA substitution. The applicants have further found that the ionexchangers of the present invention maintain their protein capacity upto modest ionic strength, about 50 mM NaCl, allowing them to be moreindustrially useful than known commercially available QA derivatisedhydroxyalkylated cross-linked regenerated cellulose anion exchangerswith a lower level of QA substitution. The latter loses capacityimmediately the ionic strength is raised above the minimum levelprovided by the dilute buffer salts, i.e. about 10 mM.

[0053] The anion exchangers of the present invention therefore haveparticular application in recovering proteins from protein-containingsolutions having more than a relatively low ionic strength, and inparticular, and surprisingly, for recovering whey proteins from milk andmilk derived process streams, such as whey and whey proteinconcentrates.

[0054] The invention will now be described in more detail with referenceto the following non-limiting examples.

EXAMPLES Example 1 Comparative

[0055] (a) Preparation of Hydroxypropyl Cellulose (HP-Cellulose)

[0056] Granular regenerated cellulose (14 g) (150-250 μm) (LifeTechnologies Ltd, Auckland, New Zealand) was mixed in a stainless steelvessel with cold 25% (w/v) aqueous sodium hydroxide (21 mL) and 0.84 mLof epichlorohydrin dissolved in 7 mL of propylene oxide. The mixture wasstirred thoroughly until the cellulose had finished swelling and all theliquid had been absorbed. The reaction vessel was then sealed and placedin a water bath at room temperature and heated to 50° C. over 30minutes. After one hour the reaction vessel was cooled and damp, friablecellulose powder (cross-linked and hydroxypropylated cellulose,HP-cellulose) was taken and, without washing, divided into seven equalfractions.

[0057] (b) Alkylation with Glycidyltrimethylammonium Chloride (GTAC)

[0058] Each of the HP-cellulose fractions was placed in a screw-toppedjar, cooled to 4° C. and mixed with an aliquot (1-4 mL) of an aqueoussolution (50% w/v) of glycidyltrimethylammonium chloride (GTAC). (Usingvolumes larger than 4 mL of this reagent did not give satisfactoryreaction mixtures or products.) The jars were sealed and placed in awater bath at 25° C. for 3 hours. The QA-cellulose products were soakedin water and then collected on sintered glass filters and washed withwater, 1 M hydrochloric acid and further water, before being drained onthe filter under vacuum.

[0059] Small samples (about 5 g) of the moist products were converted totheir hydroxide form by further washing with 1M sodium hydroxidefollowed by demineralized water. The samples were then titrated in 1Msodium chloride with 1.00 M hydrochloric acid to an end-point of pH 4.After titration each sample was collected on a dry tared sintered-glassfilter, washed with water and dried overnight at 105° C. Thesubstitution level was calculated as the small ion exchange capacity(S.I.C.) in milli-equivalents per dry gram (meq/g), i.e. S.I.C.=V/wtwhere V=volume in mL of 1.00 M HCl, and wt=dry weight of the sample (g).

[0060] Further samples in the chloride form were assayed for theirprotein binding capacities. A 0.5% solution of β-lactoglobulin wasprepared in 0.01 M sodium dihydrogen phosphate. The pH of this solutionwas adjusted to 7.5 by the careful addition of 5 M sodium hydroxide.Aliquots (20 mL) were transferred to vials containing weighed samples ofmoist QA cellulose (300-400 mg). The vials were then sealed and gentlymixed for 2 hours at room temperature. They were left to stand for 2-5minutes before a sample of the supernatant was taken and filteredthrough a 2 mL disposable column (Pierce Chemical Co. USA). A 1 mLsample of the filtrate was added to 20 μL of 1 M hydrochloric acid andmade up to 10 mL total volume with water before measuring the absorbanceat 280 nm. The dry matter of the ion exchanger used was determined bydrying samples (0.5-1 g) in triplicate. The capacity of the exchanger,grams of protein per gram of dry ion exchanger, was calculated bycomparison with an A₂₈₀ reading of the original protein solution dilutedsimilarly.

[0061] The results of these tests are shown in FIGS. 1 and 2. FIG. 1clearly indicates a maximum substitution level of 1.3-1.4 meq/g whichcan be achieved using this reagent. FIG. 2 shows the β-lactoglobulincapacity as a function of substitution level and indicates that themaximum capacity of around 2.1 g/g would not be improved by raising thesubstitution level above 1.3 meq/g.

Example 2

[0062] QA GibcoCel™ HG2 (1.17 meq/g), a commercially available anionexchanger made from the same granular regenerated cellulose as used inExample 1, was obtained from Life Technologies Ltd, Auckland, NewZealand. It was suspended in water and then collected on asintered-glass filter where it was washed with 1 M hydrochloric acid,water, 1 M sodium hydroxide and finally de-ionised water. It was thendrained of excess water by vacuum filtration. This QA cellulose in itshydroxide form was then further alkylated to raise the density ofpositively charged QA groups.

[0063] The QA GibcoCel™[OH⁻] was made up to a thick slurry by theaddition of water and 30% (w/v) aqueous sodium hydroxide. The mixturewas chilled before adding (3-chloro-2-hydroxypropyl)trimethylammoniumchloride (60 wt. % solution in water). The amounts used are shown inTable 1. These ingredients were mixed as a slurry for 17 hours at roomtemperature followed by 2 hours at 60° C. The QA cellulose products werecollected on filters and washed with water, 1 M hydrochloric acid andde-ionised water before removing the excess water by vacuum filtration.TABLE 1 Preparation Details and Properties of QA Celluloses QA GibcoCel#1 #2 #3 Preparation QA GibcoCel ™ (wet g) — 45 45 45 Water (rnL) — 3131 28.5 30% NaOH (mL) — 5 5 7.5 CHPTAC* (mL) — 3 6 9 Properties S.I.C.(meq/g) 1.17 1.51 1.84 2.02 β-1 g capacity (g/g) 1.85 1.75 1.65 1.38

[0064] Samples of each product and the starting QA GibcoCel™ wereanalysed to determine their small ion exchange capacities andβ-lactoglobulin β-lg) capacities as described in Example 1. The results,shown in Table 1, clearly indicate that the subsitution level ofquaternary amino groups on QA GibcoCel™ can be raised to 2 meq/g by thisalkylation procedure but that there is no benefit for theβ-lactoglobulin capacity under conditions that are typically used tomeasure protein capacity. In fact the capacity deteriorated,particularly for preparation #3.

Example 3

[0065] The β-lactoglobulin capacity tests on the four QA cellulosesdescribed in Example 2 were repeated in the presence of 80 mM sodiumchloride. This was achieved using the capacity test as described inExample 1 except that the β-lactoglobulin was dissolved in 0.01 M sodiumdihydrogen phosphate containing 80 mM sodium chloride and adjusted to pH7.5. The results are shown in Table 2. When the ionic strength of thetest solution was deliberately raised in this way then an increase inthe protein capacity of 50% or more was observed when the substitutionlevel of QA GibcoCel™ was raised to >1.5 meq/g. TABLE 2 β-LactoglobulinCapacities of QA Celluloses in 80 mM NaC1 at pH 7.5 Preparation QAGibcoCel #1 #2 #3 S.I.C. (meq/g) 1.17 1.51 1.84 2.02 β-1g capacity (g/g)0.64 0.98 1.10 1.11

Example 4

[0066] The effect of ionic strength on β-lactoglobulin bindingcapacities of QA GibcoCel™ (1.17 meq/g) and the QA cellulose preparation#2 (1.84 meq/g) from Example 2 was further investigated using a capacitytest similar to that outlined in Example 1 but with sodium chloridepresent at different concentrations. This was achieved as follows.

[0067] A 1% solution of β-lactoglobulin was prepared in 0.02 M sodiumdihydrogen phosphate. The pH of this solution was carefully adjusted to7.5 with 5 M sodium hydroxide. Samples of ion exchanger (300-400 mg)were weighed into glass vials. To these were added enough water and 1 Msodium chloride, together totalling 10 mL, to give final sodium chlorideconcentrations of 0, 25, 50, 75, 100 and 125 mM following the lateraddition of 10 mL of the protein solution. After mixing gently for 2hours a sample of the supernatant was taken and assayed at 280 nm asdescribed in Example 1. The results are shown in FIG. 3. In the presenceof sodium chloride at concentrations >100 mM, the β-lactoglobulincapacity of the more highly substituted QA cellulose was more thandouble that of QA GibcoCel™.

Example 5

[0068] This was similar to Example 1 except that a more concentratedsolution of glycidyltrimethylammonium chloride (GTAC) was used, 70%instead of 50%.

[0069] Granular regenerated cellulose (14 g) was converted intoHP-cellulose as described in Example 1. At the end of reaction, 28 mL of70% (w/v) GTAC was added to the chilled HP-cellulose, mixed thoroughlyand held for 3 hours at 25° C. The QA cellulose product was washed upand analysed as described in Example 1. The substitution level was 1.68meq/g and the β-lactoglobulin capacity, 2.06 g/g.

Example 6

[0070] (a) Preparation of HP-Cellulose

[0071] Granular regenerated cellulose powder (10 g) was mixed with 15 mLof cold 30% (w/v) aqueous sodium hydroxide, 0.7 mL of epichlorohydrindissolved in 5 mL of propylene oxide and reacted at 50° C. for 1 hour asdescribed in Example 1.

[0072] (b) Alkylation with (3-chloro-2-hydroxypropyl)trimethylammoniumChloride (CHPTAC)

[0073] After chilling the reaction vessel and contents, an aqueoussolution of (3-chloro-2-hydroxypropyl)trimethylammonium chloride (18 mLof 60 wt. %) was slowly added to it while stirring thoroughly. It wasthen held at 25° C. for 17 hours followed by 1.5 hours at 60° C. The QAcellulose product was soaked in excess water and collected on a filter,washed with water, 1M hydrochloric acid and then de-ionised water.Samples were analysed as described in Example 1. The substitution levelwas found to be 2.08 meq/g and the β-lactoglobulin capacity, 2.03 g/g.

Example 7

[0074] A sample of QA cellulose (2.08 meq/g) from Example 6 was furtherprocessed in place of QA GibcoCel™ (1.17 meq/g) as described in Example2 for product #2. Six mL of (3-chloro-2-hydroxypropyl)trimethylammoniumchloride was reacted with 45 g of QA cellulose [OH⁻], 31 mL of water and5 mL of 30% (w/v) aqueous sodium hydroxide. The product had asubstitution level of 2.52 meq/g and a β-lactoglobulin capacity of 1.96g/g.

Example 8

[0075] The β-lactoglobulin capacities of the QA celluloses from Examples6 (2.08 meq/g) and 7 (2.52 meq/g) were determined over a range of ionicstrengths (0-125 mM NaCl) at pH 7.5 as described in Example 4. Theresults are shown in FIG. 4 along with those for QA GibcoCel™. Althoughthe substitution level had little impact on the protein capacity in theabsence of sodium chloride, in the presence of elevated ionic strengththe more highly substituted QA celluloses showed considerably enhancedcapacities, up to four times greater at a sodium chloride concentrationof 125 mM.

Example 9

[0076] Clarified cheese whey was adjusted from pH 5.7 to 6.5 withaqueous sodium hydroxide. Aliquots (50 g) of this were then mixed atroom temperature with 10 mL (6.67 g) samples of QA GibcoCel™ and of themore highly substituted QA celluloses prepared from it (Preparations #2,#3 and #4 from Example 2). (All the QA celluloses after washing anddraining were found to have a settled volume of 1.5 mL/wet g by separateexperiment where a sample (about 10 g) was allowed to settle in water ina 25 mL measuring cylinder overnight.) After mixing for 1 hour, the QAcelluloses were separated from the protein-reduced whey on sinteredglass filters and washed with water. The combined filtrate and washings(60 g) were analysed for total nitrogen and non-protein nitrogen todetermine the residual protein concentrations. A sample of the cheesewhey at pH 6.5 was similarly analysed and the amount of protein (%)adsorbed by each of the QA celluloses calculated. The results are shownin Table 3.

[0077] Samples of the whey and the protein-reduced filtrates were alsoanalysed by reverse phase HPLC to determine the residual concentrationof β-lactoglobulin. These values were used to calculated the amount ofβ-lactoglobulin (%) absorbed from the whey. The results are shown inTable 3. Clearly it would be advantageous to use a product with asubstitution level of 1.5 meq/g or greater when recovering protein orβ-lactoglobulin from whey by ion exchange. TABLE 3 Protein Adsorptionfrom Cheese Whey by QA Celluloses Substitution Level Total Proteinβ-lactoglobulin QA Cellulose* (meq/g) Adsorbed (%) Adsorbed (%) QAGibcoCel ™ 1.17 52 56 Preparation #1 1.51 66 78 Preparation #2 1.84 7289 Preparation #3 2.02 76 93

INDUSTRIAL APPLICATION

[0078] It is believed that the anion exchangers of the presentinvention, which combine the industrial suitability of ahydroxyalkylated, cross-linked regenerated cellulose matrix with agreater protein capacity when used in the processing of milk proteincontaining raw materials, will prove particularly useful in the dairyindustry.

[0079] Although the invention has been described with reference toparticular embodiments, those persons skilled in the art will appreciatethat variations and modifications may be made without departing from thespirit and scope of the invention as defined in the claims.

What we claim is:
 1. An anion exchanger comprising a water insoluble,hydrophilic, water swellable, hydroxy(C₂-C₄)alkylated and cross-linkedregenerated cellulose, derivatised with quaternary amino (QA) groups,wherein the level of substitution of the QA groups is 1.4milliequivalents per dry gram of anion exchanger (meq/g) or greater. 2.An anion exchanger as claimed in claim 1, wherein the level ofsubstitution of QA groups is from about 1.4 to about 2.5 meq/g.
 3. Ananion exchanger as claimed in claim 2, wherein the level of substitutionof QA groups is from about 1.5 to about 2.5 meq/g.
 4. An anion exchangeras claimed in claim 3, wherein the level of substitution of QA groups isfrom about 1.7 meq/g to about 2.5 meq/g.
 5. An anion exchanger asclaimed in claim 1, wherein the cellulose is hydroxypropylatedcross-linked regenerated cellulose.
 6. A process of preparing an anionexchanger as claimed in any one of claims 1 to 5, the process comprisingthe step of reacting an anion exchanger comprising a water-insoluble,hydrophilic, water swellable, hydroxy(C₂-C₄)alkylated and cross-linkedregenerated cellulose derivatised with quaternary amino (QA) groups,wherein the level of substitution of QA groups is less than 1.4 meq/g,with an alkylating agent or agents capable of derivatising the anionexchanger with QA groups, under conditions suitable to achieve a levelof substitution of QA groups of 1.4 meq/g or greater on the anionexchanger.
 7. A process of preparing an anion exchanger as claimed inany one of claims 1 to 5, the process comprising the step of reacting awater-insoluble, hydrophilic, water swellable, hydroxy(C₂-C₄)alkylatedand cross-linked regenerated cellulose with an alkylating agent capableof derivatising the cellulose with QA groups, under conditions suitableto derivatise the cellulose and achieve a level of substitution of QAgroups of 1.4 meq/g or greater.
 8. A process as claimed in claim 7,including the additional step of further reacting the QA-derivatisedanion exchanger thus prepared with an alkylating agent capable ofderivatising the anion exchanger with QA groups to achieve a higherlevel of substitution of QA groups.
 9. A process as claimed in any oneof claims 6 to 8, wherein the alkylating agent is(3-chloro-2-hydroxypropyl)trimethylammonium chloride (CHPTAC).
 10. Aprocess as claimed in any one of claims 6 to 8, wherein the alkylatingagent is glycidyltrimethylammonium chloride (GTAC).
 11. A process asclaimed in any one of claims 6 to 10, wherein the alkylating agent isused at a concentration of 50 wt % or greater.
 12. An anion exchangerobtainable by a process as claimed in any one of claims 6 to 10.